During the chemical synthesis of multifunctional compounds, it is often necessary to use protecting groups so that selective chemical transformations can be performed. Ideally, a protecting group should allow for simple and efficient protection and subsequent regeneration of a functional group on the deprotected compound. Protecting groups which may irreversibly alter the functional group should be avoided. Moreover, the products should also be easily purified from side products generated during the synthesis or during the cleavage of the protecting group.
Triphenylchloromethane (also known as triphenylmethyl chloride and trityl chloride) and related derivatives have long been used for the protection of hydroxyl groups, amino groups and thiol groups. See Greene, T. W., Protecting Groups in Organic Synthesis, John Wiley and Sons, NY (1981). The triphenylmethyl cation is a sterically hindered electrophile. This property results in preferential reactivity of the trityl halide species with less hindered nucleophiles. In oligonucleotide synthesis, the 4,4'-dimethoxytriphenylmethyl-(4,4'-dimethoxytrityl) group and related 9-phenylxanthene-9-yl (more commonly known as pixyl) group are commonly used for the regioselective protection of the 5'-hydroxyl group of ribonucleoside and 2'-deoxyribonucleoside monomers.
Characteristics such as preferred acid lability, hydrophobic character and 5'-regioselectivity make these two triphenylmethyl derivatives the protecting groups of choice in oligonucleotide synthesis.
Alkoxy substitution of the phenyl rings is commonly used to increase the acid lability of the triphenylmethyl protecting group. Detailed investigation of trityl derivatives and the effects of substituents on acid lability have been performed. (Taunton-Rigby, A. et al., J. Org. Chem. 37:956-964 (1972). Appropriate substitution affords the 4,4'dimethoxytriphenymethyl group and the 9-phenyl-xanthene-9-yl group of optimal acid lability for current oligonucleotide synthesis applications. Additionally, several acid stable trityl derivatives which retain the desirable hydrophobic character and 5'-regioselectivity have been prepared, such as 4,4', 4"-tris(4,5-dichlorophthalimido)-trityl (Sekine, M. and T. Hata, J. Am. Chem. Soc. 106:5764-5765 (1984)); 4,4',4"-tris(levulinyloxy)trityl (Sekine, M. and T. Hata, Bull. Chem. Soc. Jpn., 58:336-339 (1985)); 4-(9-fluorenylmethyloxycarbonyl)oxy-4'4"-dimethoxytrityl (Happ, E. and C. S. Happ, Nucleosides and Nucleotides 7:813-816 (1988)); and 4-(9-fluorenylmethyloxycarbonyl)amino-4',4"-dimethoxytrityl (ibid.). All these compounds are substituted triphenylmethyl derivatives containing protected phenol(s) or protected exocyclic amino group(s). While the phenol(s) or exocyclic amino group(s) remain protected, the trityl ether bond is fairly stable to acidic conditions. Upon hydrazinolysis of the levulinyl protecting groups, hydrazinolysis of the 4,5-dichlorophthalimido protecting groups or alkali catalyzed beta-elimination of the 9-fluorenylmethyloxycarbonyl (Fmoc) group, the phenolic group(s) or exocyclic amino group(s) were all regenerated. The ether bond of the resulting trityl species can then be rapidly cleaved with mild acid. Trityl derivatives which have been previously described served strictly as protecting groups having unusual lability characteristics.
Triphenylmethyl protecting groups with long chain alkyl substituents have been prepared as tools for affinity chromatographic purification of oligonucleotides. Seliger, H. and H. H. Gortz, Angew. Chem. 93:709 (1981); Seliger, H. and H. H. Gortz, Angew, Chem. Inter. Ed. Engl. 20:683 (1981); Kwiatkowski, M. et al., Acta. Chem. Scand. B38:657 (1984); Schmidt, G. et al., Nucleosides and Nucleotides 7:795-799 (1988). By introducing triphenylmethyl protecting groups bearing long chain alkyl substituents to the 5'-hydroxyl terminus of oligodeoxynucleotides, stronger affinity of the full length DNA products for hydrophobic chromatographic supports can be achieved. Such derivatives are particularly useful for the purification of oligonucleotides of greater than sixty nucleotides in length. However, preparation of the long chain alkyl substituted triphenylmethyl derivatives and the four suitably protected synthetic monomers is difficult and labor intensive. These monomers are used for a single condensation reaction in an oligonucleotide synthesis.
In recent years, non-isotopic labeling of oligodeoxynucleotides utilizing biotin and fluorophores has become increasingly useful for the detection of DNA immobilized on solid supports (Beck, S. et al., Nucl. Acids Res. 17:5115-5123 (1989)); Takahashi, T. et al., Anal. Biochem. 179:77-85 (1989)); the immobilization of DNA to solid supports (Syvanen, A. et al., Nucl. Acids Res. 16:11327-11338 (1988)); Richardson R. W. and Gumport, R. I., Nucl. Acids Res. 11:6167-6184 (1983)); the affinity purification of DNA (Mitchell, L. G. and Merril, C. R., Anal. Biochem. 178, 239-242 (1989)); Dawson, B. A. et al., J. Biol. Chem. 264, 12830-12837 (1989)); and/or the sequencing of DNA (Beck, Ibid; Mitchell, et al., Ibid.; Smith, L. M., Nature 321:674-679 (1986)). Biotin and fluorophores have been incorporated into synthetic nucleic acid fragments by numerous chemical methods (Agrawal, S. et al., Nucl. Acids Res. 14:6227-6245 (1986); Forster, A. C. et al., Nucl. Acids Res. 13:745-761 (1985); Coull, J. M. et al., Tett. Lett. 27:3991-3994 (1986); Gibson, K. J. and Benkovic, S. J., Nucl. Acids Res. 15:6455-6467 (1987).
Chemical assembly of oligonucleotides is well established and has been performed by phosphodiester, phosphotriester, phosphoramidate, phosphoramidite and H-phosphonate methods. See Gait, M. J., Oligonucleotide Synthesis, A Practical Approach, IRL Press Inc., Oxford, England. The most common means by which oligonucleotides are produced is referred to as solid phase synthesis using the 2-cyanoethylphosphoramidites (Sinha, N. D. et al., Nucleic Acids Research, 12:4539-4557 (1984); and U.S. Pat. No. 4,725,677, issued to Millipore Corporation). Several suitably protected amino groups containing nucleoside derivatives (Haralambidis, J., et al., Nucl. Acids Res. 15:4857-4876 (1987); Gibson, K. J., Ibid., Smith, L. M. et al., Nucl. Acids. Res. 13:2399-2412 (1985)), thiol group containing nucleoside derivatives (Sproat, B. S., et al., Nucl. Acids Res. 15:4837-4848 (1987)) and 5'-terminal linkers (Agrawal, S., Ibid.; Coull, J. M. et al., Ibid.; Connolly, B. A., Nucl. Acids Res. 15:3131-3139 (1987); Blanks, R. and McLaughlin, L. M., Nucl. Acids Res. 16:2659-2669 (1988); Connolly, B. A. and Ridge, P., Nucl. Acids Res. 13:4485-4502 (1985)) have been prepared for DNA labeling applications. All of these synthons are easily incorporated into oligodeoxynucleotides during chemical assembly. All of these linkers can be further reacted to introduce a label into the oligonucleotide. The linkers, however, cannot be removed to generate a natural (unmodified) oligonucleotide.
The polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202 issued to Cetus Corporation) is a method used to exponentially amplify a nucleic acid sequence in vitro. The method uses two short oligonucleotide primers which are complementary to different strands of a DNA template and flank the region of the nucleic acid sequence to be amplified. Using a thermostable DNA polymerase and repeated cycles of template denaturing, primer annealing and primer extension, it is possible to rapidly prepare large quantities of a defined nucleic acid sequence from a few or even a single template copy. However, the flanking sequences must first be determined in order to prepare complementary primers.
Primers used in PCR will be incorporated at the 5'-hydroxyl terminus of the amplified products. If one (or both) of the primers are labeled in such a manner as not to interfere with primer annealing and primer extension, it is inevitable that the amplified product will contain the label. It would be desirable to chemically assemble oligonucleotide primers in which the label can be later removed to yield unmodified DNA.
The immobilization of PCR amplified products to a solid support has been performed by using biotin-labeled primers and a streptavidin agarose support (Mitchell, L. G., et al., Anal. Biochem. 178:239-242 (1989)). When a single labeled primer is used, it was possible to denature the immobilized double-stranded product and isolate the single-stranded sequence extended from the unlabeled primer. However, the affinity of the biotin-streptavidin complex is so great (K.sub.d is reported to be 10.sup.-15) that it is essentially impossible to remove the biotin-labeled strand from the support, thus interfering with isolation of the double-stranded product. Moreover, based on the limitations afforded by current oligonucleotide synthesis and labeling chemistries, it would be desirable to have a method in which biotin can be cleaved from the amplified DNA so that the unmodified double-stranded product can be obtained.